Readout of Histone and DNA Epigenetic Marks

Readout of Histone and DNA Epigenetic Marks


Our group has contributed to the molecular and functional understanding of histone mark and DNA methylation mark readout by writer, reader and eraser proteins, as well as investigated the crosstalk between histone and DNA methylation marks. In addition, we have also studied aspects of RNA-directed DNA methylation in plants.


Histone Mark Readout

Readout of distinctly modified histones by specialized ‘effector’ proteins, such as readers, writers and erasers, constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes.

We have written two reviews on readout of histone marks (Taverna et al. 2007; Patel & Wang, 2013) and one on combinatorial readout of histone Kme and Kac marks (Ruthenburg et al. 2007).

Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. & Patel, D. J. (2007). How chromatin-binding modules interpret histone modifications: Lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025-1040.

Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D. (2007). Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983-994.

Patel, D. J. and Wang, Z. (2013). A structural perspective of readout of epigenetic posttranslational modifications. Ann. Rev. Biochem. 82, 81-118.

Readers of Kme Marks. In collaboration with the David Allis lab (Rockefeller University), we have studied the binding H3Kme3-containing peptides to the PHD fingers and MBT repeats to identify ‘surface groove’ (PHD finger) (Li et al. 2007) and ‘cavity insertion’ (MBT domain) (Li et al. 2006) modes of methyl-lysine recognition, in each case mediated by aromatic cage capture of the methylated-lysine. These studies establish new insights into sequence-, site- and methylation state-specific readout of histone lysine methylation states and further demonstrate that dysregulation of oncogenic readers perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during mammalian development Wang et al. 2009). In parallel studies with the Or Gozani lab (Stanford University), we have identified the BAH domain as a novel methyllysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery and defining a pivotal etiologic role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

Li, H., Ilin, S., Wang, W. K., Wysocka, J., Allis, C. D. & Patel, D. J. (2006). Molecular basis for site- and state-specific readout of histone H3 lysine 4 trimethylation by NURF BPTF PHD finger. Nature 442, 91-95 (Kuo et al. 2012)

Li, H., Wang, W. K., Fischle, W., Duncan, E. M., Liang, L., Allis, C. D. & Patel, D. J. (2007). Structural basis for lower lysine methylation state-specific readout by MBT repeats and an engineered PHD finger module. Mol. Cell 28, 677-691.

Wang, G. G., Song, J., et al., Patel, D. J. & Allis, C. D. (2009). Haematopoietic malignancies initiated by dysregulation of a chromatin-binding PHD finger. Nature 459, 847-851.

Kuo, A. J., Song, J. et al., Patel, D. J. & Gozani, O. (2012). ORC1 BAH domain links emethylation of H4K20 to DNA replication licensing and Meier-Gorlin syndrome. Nature 484, 115-119.

Combinatorial Readout of Kme and Kac Marks. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into more specific biological outcomes. The PHD-Bromodomain cassette is the most frequently observed dual reader module in eukaryotes. Given that the PHD finger targets methylated-lysine marks and the bromodomain targets acetyl-lysine marks, there is a potential for combinatorial readout and synergistic impact on binding affinities. To this end, in collaboration with the Michelle Barton lab (M. D. Anderson Cancer Center), we investigated the readout by the PHD-Bromodomain cassette of the chromatin regulator tripartite motif-containing 24 (TRIM24) (Tsai et al. 2010) and in a collaborative study with the Joan Massague lab (MSKCC) the corresponding readout by TRIM33 (Xi et al. 2012). Notably, TRIM24 negatively correlates with survival of breast cancer patients, while nodal effectors use the H3K9me3 mark recognized by TRIM33 as a platform to switch master regulators of stem cell differentiation from the poised to the active state. In collaboration with the David Allis lab, we investigated the binding by the PHD-Bromodomain cassette of BPTF at the nucleosomal level to demonstrate a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells and this module colocalizes with these marks in the genome (Ruthenburg et al. 2011). Notably, in collaboration with the David Allis lab, we demonstrated the MLL1 PHD-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3 marks and cyclophilin-mediated expression through histone deacetylase (HDAC) recruitment (Wang et al. 2010).

Tsai, W-W., Wang, Z., et al., Patel, D. J. & Barton, M. C. (2010). TRIM24 links recognition of a non-canonical histone signature to breast cancer. Nature 468, 927-932.

Wang, Z., Song, J., Milne, T. A., Wang, G. G., Li, H., Allis, C. D. & Patel, D. J. (2010). Pro isomerization in MLL1 PHD3-Bromo cassette connects H3K4me3 readout to CyP33 and HDAC-mediated repression. Cell 141, 1183-1194.

Ruthenburg, A., Li, H. et al., Muir, T. W., Patel, D. J. & Allis, C. D. (2011). Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions. Cell 145, 692-706.

Xi, Q., Wang, Z. et al., Patel, D. J. & Massague, J. (2012). A poised chromatin platform for Smad access to master regulators. Cell 147, 1511-1524.

Writers of Kme Marks. Histone lysine methyltransferases such as DOT1L and MLL1 target histone tails in a sequence-, site- and methylation state-specific manner.

The evolutionarily conserved lysine methyltransferase (KMT) DOT1L methylates H3K79 and contributes to the pathogenesis of MLL-rearranged leukemia. AF10, a DOT1L cofactor, is required for maintenance of global H3K79me and cooperates with DOT1L in leukemogenesis. In collaboration with the Or Gozani lab, structure-function studies showed that recognition of H3 by the PHD finger-Zn knuckle-PHD finger (PZP) within AF10 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells (Cheng et al. 2015).

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family play crucial roles in chromatin regulation and are implicated in oncogenesis. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling methylation of H3K36. In collaboration with the Zhanxin Wang (Beijing Normal University) and Or Gozani labs, we have solved the cryo-EM structures of NSD2 and NSD3 bound to mononucleosomes, causing unwrapping of the DNA near the linker region, thereby facilitating insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA (Li et al. 2021). Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3.

Chen, S., et al., Armstrong, S. A., Patel, D. J. and Gozani, O. (2015). The PZP domain of AF10 senses unmodified H3K27 to regulate DOT1L-methylation at H3K79. Mol. Cell 60, 319-327.

Li, W. et al., Gozani, O., Patel, D. J. & Wang, Z. (2021). Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases. Nature 590, 498-503.

Erasers of Kme Marks. The Jumonji (Jmj) family of histone lysine demethylases (KDMs) are Fe2+ and alpha-ketoglutarate dependent multi-domain oxygenases that constitute essential components of regulatory transcriptional chromatin complexes and demethylate histone methylated-lysine residues in a methylation-state and sequence-specific context. In collaboration with the Or Gozani lab, our structure-function studies reveal that H3K36-specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove in the demethylase (Cheng et al. 2014).

In collaboration with the GlaxoSmithKline group, we capitalized on a structure-guided small molecule and chemoproteomics approach targeted to the H3K27me3-specific demethylase KDM6 subfamily, thereby generating the first small molecule catalytic site inhibitor that is selective for the H3K27me3 Jmj subfamily and solving its structure of the complex (Kruidenier et al. 2012).

Cheng, Z. et al., Gozani, O. and Patel, D. J. (2014). A molecular threading mechanism underlies jumonji lysine demethylase KDM2A regulation of methylated H3K36. Genes Dev. 28, 1758-1771.

Kruidenier, L. et al., Patel, D. J., Lee, K., & Wilson, W. (2012). A selective H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488, 404-408.


DNA Methylation Mark Readout

Methylation of cytosines in DNA is a stable epigenetic mark that impacts on gene regulation, imprinting, X chromosome inactivation and transposon silencing. Given that only certain CpG sites are methylated, there is currently considerable room to improve our understanding of the observed tissue- and cell-type specificity of DNA methylation. DNA methylation plays a critical role in epigenetic regulation given that defects and abnormalities in writing, reading and erasing of methylation marks are embryonic lethal in mammals and can lead to pleiotropic defects in plants.

Readers of mC DNA Marks. In a collaborative effort with the Steve Jacobsen lab, we demonstrated that the SET and RING-associated (SRA) domain of SUVH5 binds methylated DNA in all contexts to similar extents through a dual flip out mechanism. Our structure-guided in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9me2 modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci (Rajakumara et al. 2011).

Rajakumara, E. et al., Patel, D. J. & Jacobsen, S. E. (2011). A dual flip out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo. Genes Dev. 25, 137-152.

Writers of mC DNA Marks in Mammals. Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1), the major maintenance DNA methyltransferase in animals. We have solved crystal structures of mouse DNMT1 in the free state and bound to duplex DNA containing either unmethylated (Song et al. 2011) or hemimethyated CpG sites (Song et al. 2012). Our structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensure the high fidelity of DNMT1-mediated maintenance DNA methylation.

Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. (2011). Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331,1036-1040.

Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. (2012). Structure-based mechanistic insights in DNMT1-mediated maintenance DNA methylation. Science 335, 709-712.


Writers of mC DNA Marks in Plants. Domains rearranged methytransferase (DRM) is a key de novo methyltransferase in plants. In a collaborative effort with the Steve Jacobsen lab (UCLA Medical School, CA), structure-function studies establish that DRM2 homodimer is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts (Stroud et al. 2014). In a related project championed by the Steve Jacobsen lab, studies demonstrate extensive dependencies of small RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors (hong et al. 2014).

Stroud, H., Do, T., Du, J., Zhong, X., Feng, S., Johnson, L., Patel, D. J. and Jacobsen, S. E. (2014). Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64-72.

Zhong, X., Du, J. et al., Patel, D. J. and Jacobsen, S. E. (2014). Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157, 1050-1060.


Writers of mC DNA Marks in Fungi. In collaboration with the Hiten Madhani lab (Stanford University), we solved cryo-EM structures of Cryptococcus neoformans DNMT5 in three states revealing an elaborate allosteric cascade in which hemimethylated DNA first activates the SNF2 ATPase domain to bind ATP which then triggers striking structural reconfigurations of both the DNA and the methyltransferase domain to enable cofactor binding and catalysis while ejecting non-cognate DNA (Wang et al. 2022).

Wang, J. et al., Madhani, H. & Patel, D. J. (2022). SNF2 ATPase remodels DNA methyltransferase to enable durable epigenetic memory. Mol. Cell 82, 1186-1198.


Crosstalk Between Histone and DNA Marks

Both histone modification and DNA methylation contribute to the establishment of patterns of gene repression during development, highlighting interest in an improved mechanistic understanding of the crosstalk between these marks.

We have written a review in collaboration with the Steve Jacobsen lab on crosstalk between histone and DNA methylation marks (Du et al. 2015).

Du, J., Johnson, L. M., Jacobsen, S. E. and Patel, D. J. (2015). DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 16, 519-532.


Epigenetic Crosstalk in Plants. In Arabidopsis, CHG DNA methylation is controlled by the H3K9me mark through a self-reinforcing loop between DNA methyltransferase Chromomethylase3 (CMT3) and H3K9 histone methyltransferase Kryptonite (KYP). CHG methylation by maize Chromomethylase ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via aromatic cage capture by both BAH and Chromo domains, with mutations abolishing either interaction disrupting CMT3 binding to nucleosomes and showing a complete loss of CMT3 activity in vivo (Du et al. 2012). In a companion collaborative effort with the Steve Jacobsen lab, the structure was solved of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, establishing how methylated DNA recruits KYP to the histone substrate (Du et al. 2014). Together, the structures of CMT3 and KYP complexes provide insights into molecular mechanisms linking DNA and histone methylation.

Du, J. et al., Patel, D. J. & Jacobsen, S. E. (2012). Dual binding of chromomethylase BAH and chromo domains to H3K9me2-containing nucleosomes in the targeting of DNA methylation. Cell 151,167-180.

Du, J. et al., Patel, D. J. and Jacobsen, S. E. (2014). Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Mol. Cell 55, 495-504.


Epigenetic Crosstalk in Mammals. In a collaborative project with the Stephen Jane lab (Melbourne, Australia), we demonstrate that H4R3me2s formation by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation de novo DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif (Zhao et al. 2009). Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.

In a collaborative project championed by the groups of Jikui Song (Ucal-Riverside) and Greg Wang (Univ. of North Carolina), it was shown that the replication foci targeting sequence (RFTS) domain of DNMT1 is a specific reader for H3K9me3/H3Ub, defining a pathway whereby H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation (Ren et al. 2021). In a companion collaborative project championed by the groups of Jikui Song and Greg Wang, it was shown that DNMT1 specifically recognizes the H4K20me3 mark via its first bromo-adjacent-homology domain (BAH1) such that multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability (Ren et al. 2020).

Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, the Davis Allis lab applied protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs), thereby establishing that histone modification ‘reading’ and DNA methylation are closely coupled in mammalian cells (Noh et al. 2015).

Zhao, Q. et al., Patel, D. J., Allis, C. D., Cunningham, J. M. & Jane, S. M. (2009). PRMT5-mediated methylation of histone H4R3 recruits DNMT3A coupling histone and DNA methylation in gene silencing. Nat. Struct. Mol. Biol. 16, 304-311.

Noh, K. M. et al., Melnick, A, Patel, D. J., Li, H. and Allis, C. D. (2015). Engineering of a histone recognition domain in Dnmt3a alters the epigenetic landscape and phenotypic features of mouse ESCs. Mol. Cell 59, 89-103.

Ren, W. et al., Patel, D. J., Wang, G. G. & Song, J. (2020). Direct readout of heterochromatic H3K9me3 and H4K20me3 regulate DNMT1-mediated maintenance DNA methylation. Proc. Natl. Acad. Scis. USA 117, 18439-18447.

Ren, W. et al., Patel, D. J., Wang, Y., Cui, Q., Strahl, B. D., Gozani, O., Miller, K. M., O’Leary, S. E., Wade, P. A., Wang, G. G. & Song, J. (2021). DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation. Nat. Commun. 12: 2490.


RNA-directed DNA Methylation

In Arabidopsis thaliana, DNA methylation is established by Domains rearranged methyltransferase 2 (DRM2) and targeted by 24-nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis, and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors.


Pol-IV Mediated Pathway. To understand the mechanisms controlling Pol-IV targeting, we investigated the function of Sawadee homeodomain homolog 1 (SHH1), a Pol-IV-interacting protein, in collaboration with the Steve Jacobsen lab. We show that key structure-derived residues within the lysine-binding pockets involved in recognition of unmethylated K4 and methylated K9 of SHH1 are required in vivo to maintain siRNA and DNA methylation level, as well as Pol-IV occupancy at RdRM targets (Law et al. 2013).

Law, J. A., Du, J. et al., Patel, D. J. & Jacobsen, S. E. (2013). SHH1 recruits RNA polymerase-IV to RNA-directed DNA methylation targets. Nature 498, 385-389.


Pol-V Mediated Pathway. RdDM in plants depends on the synthesis of non-coding RNAs by Pol V.  In collaboration with the Steve Jacobsen lab, we report on the structure of SUVH9 which reveals a two-helix bundle that is sandwiched between the SRA and pre-SET/SET domains and an incomplete SAM binding site, as well as the absence of a peptide substrate-binding cleft, reflecting the absence of a POST-SET domain and the lack of methyltransferase activity in vitro (Johnsn et al. 2014). Our results suggest that the primary function of SUVH2/SUVH9 is in recruitment of Pol V to chromatin through its methyl-DNA binding SRA domain, providing a self-reinforcing mechanism through which DNA methylation promotes the transcription of non-coding RNAs that in turn targets further DNA methylation.

Johnson, L. M., Du, J. et al., Patel, D. J. & Jacobsen, S. E. (2014). SRA/SET domain proteins link RNA polymerase V occupancy to DNA methylation. Nature 507, 124-128.